The objective is to understand the fundamental biochemical mechanisms of virus assembly and replication. This proposal will focus on the nucleocapsid proteins of Sendai virus, a useful laboratory model of the medically important paramyxovirus family. The proposed experiments are aimed at determining the functions of each nucleocapsid protein, and exploring the relationship of protein structure to function. Three specific questions will be addressed. [i] How is virus genome RNA encapsidated by viral nucleoprotein NP? This initial assembly step is a key event in virus morphogenesis. [ii] How do viral proteins L and P combine with the encapsidated virus genome to form the functional RNA polymerase complex? Assembly of these additional proteins onto the ribonucleoprotein core of the nucleocapsid necessarily precedes viral RNA synthesis. [iii] How does the nucleocapsid complex function to synthesize viral RNA? Enzymatic activities of the complete nucleocapsid are responsible both for viral gene expression and for viral genome replication. This proposal will test the hypotheses that each nucleocapsid protein is multifunctional and that each function can be mapped to a discrete protein domain. Individual roles of each protein in assembly and enzymatic activity of the nucleocapsid complex will be dissected by generating targeted deletions or point mutations in vDNA clones representing each protein. Mutated proteins will be evaluated for the ability to participate in protein:RNA or protein:protein interactions important for nucleocapsid assembly, and in the enzymatic steps of viral RNA synthesis. Functional consequences of mutation will be evaluated both by in vitro reconstitution of assembly or transcription processes, and by exploiting a system for reverse-genetic encapsidation and expression of foreign RNA which has recently been developed in this laboratory. This combination of biochemical and reverse-genetic techniques will permit the mapping of individual activities to discrete protein regions, contributing both to the functional characterization of each nucleocapsid component and to an overall understanding of the interactions required for virus replication. The knowledge gained will be useful in devising effective strategies for the treatment or prevention of paramyxovirus diseases.